Peroxynitrite (ONOO-), a highly reactive nitrogen species produced by macrophages and other cells, is known to attach cysteine and tyrosine residues, being 2000 times more potent than H2O2 in oxidizing protein thiols. It has bactericidal activity and it is a likely mediator of some host-defense mechanisms. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an essential enzyme in the glycolytic pathway which has a critical cysteine at its active site. GAPDH, is modified covalently by NAD in the presence of nitric oxide (NO), in a reaction involving a cysteine residue. The ability of ONOO- to support this modification was examined and compared to the reactivities of nitric oxide generated from sodium nitroprusside (SNP). Effects of both agents on GAPDH activity were assessed by spectrophotometric measurement of NADH production. ONOO- inactivated GAPDH at significantly lower concentrations than did SNP and supported the covalent modification by NAD. Incubation of GAPDH with ONOO- and 14CNAD, resulted in equal incorporation of nicotinamide and adenine moieties. Replacement of [adenylate-32P]NAD by 32P adenosine 5~-diphosphoribose, decreased incorporation of radioactive label by 90%. Unlike the modification mediated by SNP, that supported by ONOO- was not sensitive to mercuric chloride (HgCl2), an agent known to cleave thiol linkages. Incorporation of label was enhanced by increasing pH, but the chemical sensitivity of the product was unchanged. GAPDH modified by NAD in the presence of SNP failed to react with anti-nitrotyrosine antibodies, but reacted strongly with ONOO- treated enzyme. These data support the hypothesis that ONOO- supports the modification of GAPDH by NAD, but the reaction involves an amino acid other than cysteine, based on the chemical sensitivity of the product.